The steroid hormone ecdysterone is known to be intimately involved in the regulation of many developmental processes in Drosophila melanogaster. By attempting to identify newly induced interactions occurring between the DNA of responsive genes and trans acting factors activated in the presence of hormone, this proposal seeks to initiate a molecular description of how this hormonal regulation occurs, and at what level a differential stage and tissue specific response from various genes is established. Drosophila possess four small heat shock genes which, in addition to being heat shock inducible, are developmentally regulated by ecodysterone. A high resolution nuclear "footprinting" technique will be applied to hormonally responsive tissue culture cells to enable putative cis regulatory elements for each of these genes to be identified by virtue of their altered accessibility on hormonal induction. Ecdysterone mediated DNA-protein interactions at these elements will lead to a characteristic footprint. A direct functional test for ecdysterone regulation via these sequences will be provided by experiments that fuse synthetic copies of identified sequences to a suitable test gene (Beta-galactosidase), followed by assays in transfected cell lines or transformed flies. The availability of cell lines lacking hormone receptor, and fly strains conditional for ecdysterone responsiveness, will enable strong selection to be applied in testing of hormonally responsive elements. Once identified, elements can be assayed for their function as tissue specific enhancers. Attempts will be made to impose DNA or chromatin structural constraints that will impair the function of these elements, thereby revealing possible modes of action. As hormonally responsive elements are confirmed, fractionation of cellular extracts, using sensitive DNA-protein binding assays should lead to purification of specific trans acting components. With the identification of further genes that display differential responses to hormone, these approaches may help provide a molecular explanation of the pleiotropic regulatory pathways mediated by ecdysterone. The combined use of genetic and molecular approaches in the Drosophila system make it an attractive one for studies of hormonal regulation. This is likely to be of particular value given that steroid hormonal regulation is a fundamental physiological process in many cells of higher eukaryotes.